The discovery of the XIST gene represents a major breakthrough in the elucidation of the phenomenon of X chromosome inactivation. A new observation from our laboratory regarding replication timing of XIST appears to have significant implications for understanding X inactivation, as well as for genes subject to genomic imprinting. We have observed that while the expressed XIST gene on the inactive X chromosome in fibroblasts replicates late (in the second half of S), its silent allele on the active X replicates relatively early in S. This is the first clear demonstration of a reverse replication timing pattern for expressed versus silent alleles of a gene. The silent allele of XIST is replicating amidst flanking expressed genes and we propose that a previously unknown repression mechanism may be involved. Our preliminary evidence suggests that repression of the duplicated, silent XIST allele may be affected by sister chromatid cohesion. We suspect that this previously unconsidered mechanism for repression control may also affect genes subject to either X chromosome inactivation or genomic imprinting. An in situ hybridization electron microscope study using gold-labeled probes is proposed to explore this possibility. We have also observed that the replication of the expressed allele of XIST can be shifted to an early-replicating pattern that is similar to that of the silent, active X allele by treatment of cells with the demethylating agent, 5-azacytidine. Preliminary evidence suggests that the methylated sites affected may be within a locus control region for the XIST domain and a study is proposed to test this hypothesis and identify the controlling sequence. This putative control locus is likely to be a critical target for X inactivation control mechanisms. A related study is based on our finding that the expressed allele of the fragile X gene, FMR1, also replicates in the second half of S. In fact, FMR1 may have been the first example of this type of gene in normal human cells. The FMR1 and XIST genes are located with heterochromatic-like regions and may represent a class of mammalian genes that are analogous to Drosophila genes that require an unusual transcriptional environment. Other late-replicating, expressed mammalian genes are likely to exist and we have developed a selection system to detect and isolate them. To determine whether such genes require unusual transcriptional conditions, we propose to examine them for cell cycle-dependence of transcription and for cell cycle-dependence of nuclease sensitivity within the promoter region. The health relatedness of this project derives from the fact that the two genes at the basis of this study, FMR1 and XIST, are critical to normal mental and sexual development.